Safety and Immunogenicity of a ChAd155-Vectored Respiratory Syncytial Virus Vaccine in Infants 6–7 Months of age: A Phase 1/2 Randomized Trial

Abstract Background Respiratory syncytial virus (RSV) is a common cause of lower respiratory tract infections in infants. This phase 1/2, observer-blind, randomized, controlled study assessed the safety and immunogenicity of an investigational chimpanzee-derived adenoviral vector RSV vaccine (ChAd155-RSV, expressing RSV F, N, and M2-1) in infants. Methods Healthy 6- to 7-month-olds were 1:1:1-randomized to receive 1 low ChAd155-RSV dose (1.5 × 1010 viral particles) followed by placebo (RSV_1D); 2 high ChAd155-RSV doses (5 × 1010 viral particles) (RSV_2D); or active comparator vaccines/placebo (comparator) on days 1 and 31. Follow-up lasted approximately 2 years. Results Two hundred one infants were vaccinated (RSV_1D: 65; RSV_2D: 71; comparator: 65); 159 were RSV-seronaive at baseline. Most solicited and unsolicited adverse events after ChAd155-RSV occurred at similar or lower rates than after active comparators. In infants who developed RSV infection, there was no evidence of vaccine-associated enhanced respiratory disease (VAERD). RSV-A neutralizing titers and RSV F-binding antibody concentrations were higher post–ChAd155-RSV than postcomparator at days 31, 61, and end of RSV season 1 (mean follow-up, 7 months). High-dose ChAd155-RSV induced stronger responses than low-dose, with further increases post–dose 2. Conclusions ChAd155-RSV administered to 6- to 7-month-olds had a reactogenicity/safety profile like other childhood vaccines, showed no evidence of VAERD, and induced a humoral immune response. Clinical Trials Registration. NCT03636906.

Background.Respiratory syncytial virus (RSV) is a common cause of lower respiratory tract infections in infants.This phase 1/2, observer-blind, randomized, controlled study assessed the safety and immunogenicity of an investigational chimpanzeederived adenoviral vector RSV vaccine (ChAd155-RSV, expressing RSV F, N, and M2-1) in infants.
Results.Two hundred one infants were vaccinated (RSV_1D: 65; RSV_2D: 71; comparator: 65); 159 were RSV-seronaive at baseline.Most solicited and unsolicited adverse events after ChAd155-RSV occurred at similar or lower rates than after active comparators.In infants who developed RSV infection, there was no evidence of vaccine-associated enhanced respiratory disease (VAERD).RSV-A neutralizing titers and RSV F-binding antibody concentrations were higher post-ChAd155-RSV than postcomparator at days 31, 61, and end of RSV season 1 (mean follow-up, 7 months).High-dose ChAd155-RSV induced stronger responses than low-dose, with further increases post-dose 2.
Respiratory syncytial virus (RSV) is a common, contagious pathogen that causes respiratory tract infections (RTIs) in people of all ages.These typically occur in autumn and winter months in temperate regions and in rainy seasons or throughout the year in the tropics [1,2], although seasonality has been impacted by the coronavirus disease 2019 (COVID-19) pandemic [3][4][5][6].RSV is one of the most common etiologies of lower respiratory tract infections (LRTIs), such as bronchiolitis and pneumonia, in infants and young children [7][8][9][10].
Two monoclonal antibodies are available for RSV-LRTI prevention [11,12], but no pediatric RSV vaccine has been licensed yet.The development of RSV vaccines was slowed down by the failure of an investigational formalin-inactivated vaccine (FI-RSV) in young children >50 years ago [13].This vaccine did not protect against RSV infection and was associated with severe respiratory illness upon natural RSV infection (particularly in young RSV-naive infants), a phenomenon called vaccine-associated enhanced respiratory disease (VAERD) [13][14][15][16][17].When evaluating new pediatric RSV vaccine candidates, it is critical to show the absence of VAERD after vaccination of RSV-naive children.
Our study evaluated an investigational RSV vaccine based on a chimpanzee-derived replication-deficient adenoviral vector (ChAd155-RSV).The vector encodes 3 recombinant proteins that are highly conserved among the 2 RSV subtypes (RSV-A and RSV-B): the surface fusion (F) protein (which elicits neutralizing antibodies), the internal nucleocapsid (N) protein, and the transcription antitermination (M2-1) protein (both sources of T-cell epitopes) [13,18].In a phase 1 study in 18to 45-year-old RSV-seropositive adults, ChAd155-RSV had an acceptable safety profile and was immunogenic [19].In a phase 1/2 study in 12-to 23-month-old RSV-seropositive children, ChAd155-RSV induced neutralizing antibodies and was well tolerated, with no evidence of VAERD upon natural infection [20].Our study evaluated the safety and immunogenicity of ChAd155-RSV administered to healthy 6-to 7-month-old infants, most of whom were seronaive.

Study Design and Participants
This phase 1/2, observer-blind, randomized controlled study was performed from 8 April 2019 to 22 July 2021 in 13 countries (Supplementary Table 1).We enrolled healthy, full-term, 6-to 7-month-old infants with no history of confirmed RSV disease or highly compatible clinical manifestations, after obtaining written informed consent from their parents or legally authorized representatives (LARs) (Supplementary Methods).
Infants were randomized 1:1:1 (Supplementary Methods) to 3 groups: RSV_1D, RSV_2D, or comparator (Figure 1).The RSV_1D group received 1 low ChAd155-RSV dose (1.5 × 10 10 viral particles) on day 1 and placebo on day 31.The RSV_2D group received 2 high ChAd155-RSV doses (5 × 10 10 viral particles) on days 1 and 31.The comparator group received either placebo only (days 1 and 31), or placebo and 1 of 4 possible active comparator vaccines depending on the country.Four-component meningococcal serogroup B vaccine (4CMenB, Bexsero, GSK) or meningococcal serogroups A, C, W, Y tetanus toxoid conjugate vaccine (MenACWY-TT, Nimenrix, Pfizer) was given on day 1, followed by placebo on day 31; pneumococcal nontypeable Haemophilus influenzae protein D conjugate vaccine (PHiD-CV, Synflorix, GSK) or meningococcal serogroups A, C, W, Y CRM 197 conjugate vaccine (MenACWY-CRM, Menveo, GSK) were given on day 31, with placebo on day 1.First doses were given before the start of the first RSV season.Infants were followed for approximately 2 years until the end of the second RSV season.RSV seasons were defined based on epidemiological data from before the COVID-19 pandemic (Supplementary Methods).To complete the active comparator vaccine schedules in the comparator groups, children received 4CMenB, MenACWY-TT, or PHiD-CV on day 61 and at the end of the first RSV season (with placebo on day 121), or MenACWY-CRM at the end of the first RSV season (with placebo on day 61).To maintain blinding, children in the ChAd155-RSV groups received 3 doses of 4CMenB, MenACWY-TT, or PHiD-CV or 2 doses of MenACWY-CRM at these same visits (Figure 1).The study was observer-blinded until day 61 and single-blinded thereafter (Supplementary Methods).
The study (ClinicalTrials.gov:NCT03636906) was conducted according to Good Clinical Practice guidelines, the Declaration of Helsinki, and applicable regulatory requirements.The study sites' institutional review boards or independent ethics committees approved the protocol and its amendments (available on https://www.gsk-studyregister.com/en/trial-details/?id=204894).An internal safety review committee and independent data monitoring committee (IDMC) monitored the participants' safety.

Objectives
The primary objective was to evaluate the safety and reactogenicity of 1 or 2 ChAd155-RSV doses until 60 days post-dose 1. Secondary objectives included safety analyses until the end of the second RSV season and evaluation of the occurrence of RSV-RTI and RSV-LRTI until the end of the second RSV season in infants who were considered RSV seronaive at screening (see "Serological Assessments" for a definition of RSV seronaive).Humoral immunogenicity until the end of the first RSV season was also evaluated as a secondary objective.Objectives are detailed in the Supplementary Methods.

Safety Assessments
The infants' parents/LARs recorded solicited adverse events (AEs) occurring within 7 days and unsolicited AEs occurring within 30 days postvaccination on diary cards.Serious AEs (SAEs) and AEs leading to study withdrawal were recorded throughout the study.Spontaneous or excessive bleeding was monitored as an AE of specific interest (AESI) (Supplementary Methods).Because of the risk of VAERD with the FI-RSV vaccine [14][15][16][17], RSV-LRTIs occurring throughout the study were considered as AESIs.Identification of RSV-LRTIs in the context of AESI reporting was based on the investigators' clinical judgment considering clinical history, examination, relevant medical evaluation, and results of a locally available diagnostic RSV test.Not all RSV-LRTIs identified by the investigators and reported as AESIs necessarily met the predefined RSV-LRTI case definition used for surveillance (see "RTI Surveillance" and Supplementary Table 2).

RTI Surveillance
Participants were monitored for RTIs and episodes of difficulty breathing or wheezing through passive and active surveillance contacts by phone or email throughout the study (Supplementary Figure 1).Study staff contacted the infants' parents/LARs (or designated persons) weekly during the RSV season or monthly outside the RSV season.Parents/LARs/ designated persons were reminded to contact the study staff when their children experienced new or worsened RTI symptoms (cough, rhinorrhea, or nasal obstruction) or episodes of difficulty breathing or wheezing.If RTI symptoms developed, an assessment visit was scheduled (Supplementary Figure 1).During this visit, study staff evaluated clinical signs and symptoms of the RTI, measured oxygen saturation and respiratory rate, and collected nasal swabs for RSV-A/RSV-B detection using quantitative reverse-transcription polymerase chain reaction (PCR) (Supplementary Methods).During the RSV season, nasal swabs were also collected monthly from all infants to detect asymptomatic RSV-RTI.To support the safety review of potential VAERD cases, a respiratory viral panel was performed (by multiplex PCR) on all RSV-positive specimens and all LRTIs matching the case definition to evaluate coinfection.Case definitions for RSV-RTI, RSV-LRTI, and (very) severe LRTI were based on those proposed by the World Health Organization (WHO) (Supplementary Table 2) [21].

Serological Assessments
Blood samples (2.5 mL) for immunogenicity analyses were drawn from all infants at screening (baseline), day 31, day 61, and at the end of the first RSV season.RSV-A neutralizing titers were measured using a neutralization test with an assay cutoff (lower limit of quantification) of 18 estimated dilution 60 (ED 60 ) [22].RSV F-binding immunoglobulin G (IgG) antibodies were measured using an enzyme-linked immunosorbent assay [22].
For the analyses in RSV-seronaive infants, a baseline RSV-seronaive status was defined as an RSV-A neutralizing titer <63.086ED 60 in the baseline sample.This cutoff was determined through modeling of data from an epidemiological study [23] and represents an RSV-A neutralizing titer 2-fold higher than the estimated titer in 6-month-olds due to residual maternal antibodies.Infants with titers equal to or above this value were considered to have experienced an RSV infection.

Statistical Analysis
The sample size (target: 150 infants; 50 per group) was based on the minimum number of infants needed to detect a VAERD signal with a magnitude like that observed in a study with the FI-RSV vaccine, in which 80% of RSV infections required hospitalization [14] (Supplementary Methods).
For all analyses, data from the different RSV_1D and RSV_2D groups (receiving the various active comparator vaccines/placebo, Figure 1) were pooled into a single RSV_1D and a single RSV_2D group.Data from the comparator groups were either pooled across all active comparators and placebo (comparator group) or across active comparators only (active comparator group, used for the analysis of solicited AEs).
Safety and reactogenicity were analyzed on the exposed set (all infants receiving ≥1 dose of ChAd155-RSV, active comparator, or placebo).The main analyses of RTI and LRTI were done on the exposed set of baseline RSV-seronaive infants.To evaluate the risk of VAERD, we assessed the proportion of baseline RSV-seronaive infants who received ChAd155-RSV, subsequently became infected with RSV, and had a very severe RSV-LRTI (per case definition, Supplementary Table 2).A risk of VAERD like that with the FI-RSV vaccine (for which 80% of RSV infections required hospitalization [14]) was considered unlikely if the upper limit of the 2-sided 90% confidence interval (CI) for this proportion was <80%.In a post hoc analysis, we calculated the relative risk of RSV-LRTI of any severity (per case definition) in a pooled RSV_1D/RSV_2D group compared to the comparator group for baseline RSV-seronaive infants.These analyses were performed on RSV-LRTIs collected until the end of the first RSV season.
The primary immunogenicity analysis was done on the perprotocol population (all vaccinated infants who complied with eligibility criteria and study procedures and had immunogenicity results available).Results focused on here are for baseline RSV-seronaive infants in the per-protocol population.Geometric mean titers (GMTs), geometric mean concentrations, and geometric mean ratios (GMRs) of postvaccination versus baseline titers/concentrations were calculated.For values below the assay cutoffs, values of half the cutoffs were used.
All analyses were descriptive and were done using SAS software version 9.4 (SAS Institute, Cary, North Carolina).
None of the RSV infections progressed to very severe RSV-LRTI.The 90% CI for the risk that ChAd155-RSV induced very severe LRTI was 0.0-14.6% in RSV_1D and 0.0-17.1% in RSV_2D recipients (based on 19 RSV infections in RSV_1D and 16 in RSV_2D at the end of RSV season 1).Additionally, the relative risk of RSV-LRTI in the pooled ChAd155-RSV versus the comparator group was 0.73 (95% CI, .22-2.47) at the end of RSV season 1.
Abbreviations: CI, confidence interval; LRTI, lower respiratory tract infection; RSV_1D, group receiving 1 low chimpanzee-derived replication-deficient adenoviral vector RSV vaccine (ChAd155-RSV) dose as dose 1 and placebo as dose 2; RSV_2D, group receiving 2 high ChAd155-RSV doses as dose 1 and 2; comparator, group receiving either placebo as dose 1 and 2, or active comparator vaccine as dose 1 or 2 and placebo as the other dose (pooled); RTI, respiratory tract infection.a All confirmed RSV infections based on central reverse-transcription polymerase chain reaction testing.
b This included both upper and lower respiratory tract infections.c Confirmed RSV infection and hospitalized for acute medical condition.
groups than in the comparator group (Table 3, Figure 4).One dose of the high-dose formulation induced a greater RSV-A neutralizing response than the low-dose formulation (GMRs of 4.44 [RSV_2D] and 2.88 [RSV_1D] at day 31 vs baseline; Table 3).The second ChAd155-RSV dose further increased the RSV-A neutralizing GMTs at day 61, with a GMR (vs baseline) of 11.80 (Table 3, Figure 4).RSV-A neutralizing GMTs were higher at the end of RSV season 1 than at day 61 in the RSV_1D and comparator groups and similar at these 2 timepoints in the RSV_2D group (Table 3, Figure 4).
Similar results were obtained in the per-protocol population (including both RSV-seronaive and non-seronaive infants) (Supplementary Table 7).

DISCUSSION
This randomized phase 1/2 study showed that administration of the investigational ChAd155-RSV vaccine to 6-to 7-monthold, mostly RSV-seronaive infants had a reactogenicity and safety profile like that of other childhood vaccines, with no indication of VAERD, and induced a humoral immune response.Our findings were in line with those of the first-in-human study with ChAd155-RSV in adults [19] and the phase 1/2 study in 12-to 23-month-old RSV-seropositive children [20].
Most solicited AEs after ChAd155-RSV vaccination were transient, of mild or moderate intensity, and were reported at similar or lower rates than in the active comparator group.Solicited AEs occurred at comparable rates in the 2 ChAd155-RSV groups, except for fever, which was more common in the RSV_2D than the RSV_1D group but occurred at a similar rate as after 4CMenB vaccination, which was the most pyrogenic comparator.The reported unsolicited AE rates were similar across study groups, and the types of AEs were consistent with usual illnesses occurring in infants.One case of gastrointestinal hemorrhage occurred, which was judged by the investigator as possibly ChAd155-RSV related.The IDMC raised no safety concerns, and the sponsor considered the event as unrelated to vaccination based on clinical and laboratory evidence and a more probable alternative etiology.SAEs occurred more frequently in the ChAd155-RSV groups (10.8% and 15.5%) than the comparator group (4.6%).The difference was mostly due to a higher rate of infections in the ChAd155-RSV groups, but no common diagnosis or etiology could be found among these infections.Interpretation of this imbalance is limited by the study's small sample size.There was no evidence to suggest that ChAd155-RSV vaccination in RSV-seronaive 6-to 7-month-old infants induced VAERD after natural RSV infection.None of the RSV infections in ChAd155-RSV-vaccinated seronaive participants progressed to very severe RSV-LRTI, and the relative risk (0.73) of RSV-LRTI in the pooled ChAd155-RSV group versus the comparator group suggests that an increase in RSV-LRTI in vaccinated infants was unlikely (although the CI around the relative risk was wide).The mechanisms that lead to VAERD after FI-RSV vaccination are not fully understood but may involve suboptimal humoral immune responses, absence of cellular cytotoxic responses, and a Th2-biased cellular response [24,25].Chimpanzee-derived adenoviral vector-based vaccines induce a humoral and cellular immune response [26], which would reduce the risk of VAERD.Preclinical studies in animal models showed that ChAd155-RSV was immunogenic and efficacious, with no signs of VAERD after RSV challenge.
A single vaccination with both the low-dose and high-dose ChAd155-RSV formulations increased RSV-A neutralizing titers and RSV F-binding IgG levels.The high-dose formulation elicited a stronger response than the low-dose formulation, and the second ChAd155-RSV dose induced a further increase in antibody levels.Antibody levels in both ChAd155-RSV groups remained above baseline levels and above levels in the comparator group at the end of the first RSV season.Natural RSV infection over the course of the season appeared to boost immunity, as evident from increased antibody levels observed in the RSV_1D and comparator groups.
The rates of RSV infection, RSV-RTI, and RSV-LRTI were numerically lowest in the RSV_2D group and highest in the comparator group over the 2 RSV seasons.Nevertheless, vaccine impact on RSV illness seemed low compared to that of the monoclonal antibody nirsevimab [27] and a maternal RSV-prefusion F candidate vaccine [28,29].However, our study was not powered (sample size too low) to draw conclusions on ChAd155-RSV vaccine efficacy.ChAd155-RSV expresses a wild-type version of the F protein with a deletion of the transmembrane domain.Experiments in cell culture indicated that the protein expressed by ChAd155-RSV is present in both the prefusion and postfusion conformation (unpublished data).Prefusion F has been shown to induce a stronger neutralizing response than postfusion F and may be the preferred vaccine antigen [30,31].
During the COVID-19 pandemic, mRNA and adenoviral vector vaccines have emerged as promising efficacious and safe alternatives to conventional vaccine approaches.These vaccine platforms allow rapid development and affordable scalability and could be readily adapted to new strains or pathogens [32].Concerns have been raised that vaccination with adenoviral vectors may induce antivector immunity, which may reduce vaccine efficacy after future readministration of the same vector [33,34].Heterologous prime-boost approaches with different adenoviral vector types or different vaccine platforms (eg, adenoviral vector followed by mRNA) might circumvent antivector immunity when booster doses are needed to provide durable protection against a pathogen [34][35][36][37][38].Further improvements of the adenoviral vector platform and future research will shed light on whether the same adenoviral vectors can be administered effectively for different pathogens during a person's life.
A limitation of the study is the restricted racial diversity of the study population (with almost no Black or Asian infants), which limits the generalizability of our results.Additionally, we did not evaluate the administration of 2 low ChAd155-RSV doses and did not analyze cell-mediated immunity.The study was impacted by its partial overlap with the COVID-19 pandemic, when circulation of RSV and other respiratory viruses was reduced, likely as a result of nonpharmaceutical interventions (eg, lockdowns, physical distancing, and masks) implemented to curb the spread of COVID-19 [3][4][5][6].This was most obvious in the second RSV season in our study, when almost no RSV infections were reported.However, the incidence of RSV infection in the first season was high enough for our analysis of the risk of VAERD to be conclusive.
In conclusion, this study showed that the investigational ChAd155-RSV vaccine administered to 6-to 7-month-old, mostly seronaive infants had a reactogenicity and safety profile that was similar to that of other childhood vaccines, with no evidence of VAERD.ChAd155-RSV elicited a neutralizing response that was higher with the high-dose than the low-dose formulation and increased after a second dose.However, development of ChAd155-RSV has been discontinued because the target efficacy profile was unlikely to be met.M. M.-P., M. R., N. V., S. N. F., T. D. L., T. I., T. P., V. N., W. P., X. S.-L., and X. N. performed the study.B. S. C., D. F., E. J. A., E. K., F. M.-T., Y. Z., I. D., J. D. C., J. M. L., J. M. M. A., M. R., N. V., S. N. F., S. K. S., T. I., T. P., V. N., W. P., X. S.-L., and X. N. were involved in the analyses or interpretation of the data.

Figure 2 .
Figure 2. Disposition of participants.a The comparator group included 22 infants who received placebo only and 43 who received active comparator and placebo (29 received 4-component meningococcal serogroup B vaccine, 1 received meningococcal serogroups A, C, W, Y tetanus toxoid conjugate vaccine, 1 received pneumococcal nontypeable Haemophilus influenzae protein D conjugate vaccine, and 12 received meningococcal serogroups A, C, W, Y CRM 197 conjugate vaccine).bRSV-seronaive at screening, ie, RSV-A neutralizing titer <63.086 estimated dilution 60, which represents a titer 2-fold higher than the estimated RSV-A neutralizing titer in infants at 6 months of age due to residual maternal antibodies (based on modeling estimates).Abbreviations: comparator, group receiving either placebo as dose 1 and 2, or active comparator vaccine as dose 1 or 2 and placebo as the other dose (pooled); RSV, respiratory syncytial virus; RSV_1D, group receiving 1 low chimpanzee-derived replication-deficient adenoviral vector RSV vaccine (ChAd155-RSV) dose as dose 1 and placebo as dose 2; RSV_2D, group receiving 2 high ChAd155-RSV doses as dose 1 and 2; (S)AE, (serious) adverse event.